A significant medical problem, namely thromboembolism, is presented by the occlusion of blood vessels due to the presence of thrombi (blood clots). Such thromboembolisms are potentially fatal, often affecting organs such as the heart or lungs. This blockage of normal blood circulation may lead to irreversible tissue damage as metabolizing cells are deprived of oxygen, nutrients and a path for disposal of waste products. A thromboembolytic event, as described above may, for example, result from the formation of a blood clot in an arterioschlerotically altered artery. Such a clot may loosen and circulate to smaller arteries until it reaches an artery or arteriole too small to permit passage of the clot. The clot thereby blocks this small artery and prevents or inhibits the passage of blood therethrough.
The restoration of normal blood flow through such clot-blocked blood vessels, particularly in time to prevent tissue damage, is an important curative goal of modern medical practice. Another medical approach to the thromboembolytic problem is to prevent the formation of vascular blood clots when conditions known to result in such clot formations are known or suspected to exist.
Certain thrombolytic agents facilitate the in vivo lysis or dissolution of blood clots. One of these agents is the bacterial protein streptokinase.
Streptokinase, a protein produced and secreted by hemolytic streptococci, was discovered and shown to cause lysis of clots containing human fibrinogen by Tillett and Garner (J. Exp. Med. 58: 485 [1933]). While the mechanism of such fibrinolysis is not totally understood, streptokinase itself is believed to bind and activate human plasminogen, the fibrinolytically inactive precursor of plasmin. The activation of such plasminogen results in the appearance of plasmin, a fibrinolytic enzyme which hydrolyzes clot fibrinogen (Castellino TIBS, p. 1 [Jan. 1979]).
Streptokinase is a thrombolytic agent currently being used for the lysis of intravascular thrombi in pulmonary or peripheral blood vessels (Marder, Ann. Intern. Med. 90: 802 [1979]). Recent studies of intracoronary administration of streptokinase in the treatment of acute myocardial infarction patients indicate a beneficial effect on the early course of acute myocardial infarction (Khaja et al. N.E. J. Med. 308, p. 1305 [1983] and Anderson et al. N.E. J. Med. 308, p. 1312 [1983]).
Streptokinase from serological group C Streptococci has been purified for clinical usage and studied by several investigators. The primary source of this streptokinase has been the culture fluid resulting from the growth of the beta-hemolytic group C organism Streptococcus equisimilis. This culture fluid contains, in addition to streptokinase (a single-chain protein with a molecular weight of 45,000 to 50,000), a number of other extracellularly secreted substances. Since many of these other substances are toxic or potentially toxic to humans, the preparation of streptokinase from culture fluid for clinical usage involves multiple steps which must be performed with great care to ensure a clinically acceptable product.
The c-DNA sequences of mammalian urokinase and tissue-type plasminogen activator have been cloned previously (Pennica et al., Nature 301, p. 214 [1983] and Ratzkin et al., Proc. Nat. Acad. Sci. 78, p. 3313 [1981]).
A publication by Sako et al. (Mol. Gen. Genet. 190, p. 271 [1983]) describes the cloning of a staphylokinase-producing Escherichia coli transformant. A polydeoxynucleotide sequence sak coding for staphylokinase, which is a plasminogen activating protein having a size of about 15 kd, was excised from a native staphylococcal-residing bacteriophage (P.phi.-2)and ligatively inserted into plasmid pBR322. This sak-modified plasmid was used to transform Escherichia coli K12 and the resultant staphylokinase-producing Escherichia coli transformant was found to secrete amounts of staphylokinase into a culture medium.